The insulin-like growth factors (IGF-I and -II) are polypeptides of approximately 70-residues with auto- and paracrine effects on cell proliferation, migration and differentiation. The IGFs binds to the IGF-1 receptor (IGFR), but a series of six binding proteins, IGFBP-1 to −6, are able to sequester IGF from IGFR because of higher affinities for IGF-I and -II. However, bioactive IGF can be released from such complexes by means of proteolytic cleavage of the binding protein, causing the generation of IGFBP fragments with diminished affinity for IGF. Specific, limited proteolysis represents the principal mechanism of IGF activation.
Several lines of evidence have demonstrated that the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) functions in the IGF system. PAPP-A specifically cleaves IGFBP-4 and IGFBP-5, thereby releasing sequestered IGF or causing binding protein inactivation. PAPP-A knockout mice are proportional dwarfs, reduced to a body mass of 60% compared to wild type littermates. This phenotype is similar to the phenotype of IGF-II knockout mice, supporting the hypothesis that IGF-II activity requires PAPP-An activity in early fetal development with cleavage of IGFBP-4 being responsible for the final delivery of IGF to the receptor. Postnatally, PAPP-A and IGFBP-4 appears to be implicated in many different processes of cell proliferation, such as wound healing, follicle selection, implantation, myoblast proliferation and differentiation, and bone formation in vivo. PAPP-A is produced by vascular smooth muscle cells following angioplasty, suggesting that it promotes neointimal cell proliferation, and it has been shown to be a serum marker of acute coronary syndromes, most likely because it is abundantly synthesized in unstable atherosclerotic plaques.
The 400 kDa PAPP-A has two subunits of 1547 residues, and it belongs to the metzincin superfamily of metalloproteinases. A laminin G-like module of unknown function is present N-terminal to the proteolytic domain, and five complement control protein (CCP) modules, enabling PAPP-A to bind to the cell surface, are located in the C-terminal end of the subunit (FIG. 1). Additionally, PAPP-A contains three Lin12-Notch repeat (LNR) modules, which are unique to PAPP-A, its homologue PAPP-A2, and the family of Notch receptors. In PAPP-A and PAPP-A2, two LNR modules (LNR1 and 2) are inserted into the proteolytic domain, whereas the third (LNR3) is located C-terminal to the CCP modules, (FIG. 1). Within the PAPP-A dimer, the LNR modules probably form trimeric units composed of LNR1 and 2 from one subunit and LNR3 from the other subunit. Compromised LNR functionality renders PAPP-A unable to cleave IGFBP-4, while cleavage of IGFBP-5 is unaffected.
The IGFs are involved in both normal physiology and human disease, e.g. cancer and cardiovascular disease, and therefore, strategies for the direct inhibition of IGF signalling are being developed. However, specific inhibition of growth promoting proteolytic activity may represent a valuable alternative, in particular because such inhibition of IGF receptor stimulation is unlikely to interfere with insulin signalling and other aspects of normal physiology.
The principle of exosite inhibition is highly relevant for many proteolytic enzymes involved in human disease, in particular multidomain enzymes such as the large group of matrix metalloproteinases (MMPs) and related enzymes. Inhibition by targeting a substrate binding exosite has at least two advantages compared to direct active site inhibition: specificity and selectivity. First, an inhibitor directed towards an exosite is less likely to influence the activity of other related proteinases with similar active site architecture. Second, cleavage of different substrates of a given proteinase may not use the same exosite and therefore can be differentially inhibited.
The literature contains many examples of proteolytic inhibition by exosite targeting. For example, MMP-2 cleavage of type I gelatin and type IV collagen has been inhibited by targeting the collagen binding domain using a synthetic peptide, and a substrate binding pocket distinct from the catalytic site of beta-APP cleaving enzyme has been targeted by synthetic peptides. Also, many exosite inhibitors have been developed towards the coagulation enzyme Factor VIIa, and although specific for this proteinase, all of its several biological substrates are targeted by such inhibitors. Therefore, to the best of our knowledge, the inhibition of IGFBP-4 proteolysis by PAPP-A represents the first example of selective inhibition of one natural proteinase substrate by exosite targeting.